The long range objective of this research is to understand how pathogenic bacteria display and utilize surface attached proteins during infections, and to use this knowledge to develop a therapeutically useful anti-infective agent. Surface proteins are frequently required for virulence, as they promote bacterial adhesion, resistance to phagocytic killing, host cell invasion, and nutrient acquisition. In gram-positive bacteria, surface proteins are covalently anchored to the cell wall peptidylglycan by sortases, a large family of cysteine transpeptidases. Research in this proposal will study the sortase enzymes and cell wall attached proteins in Staphylococcus aureus, the leading cause of hospital-acquired infections in the United States. In aim #1, we will study how the prototypical Sortase A protein (SrtA) recognizes the universally conserved LPXTG sorting signal, by solving the NMR structure of its covalent complex with a sorting signal analogue. In aim #2, we will explore how active site variations in SrtA control the sorting signals that it can recognize. This will be accomplished by determining the substrate specificities of rationally engineered single amino acid mutants. In aim #3, we will attempt to develop an inhibitor of this important enzyme class, by conducting structure activity relationship analyses on four small molecule inhibitors of SrtA that we have identified through compound library screening and rational design approaches. In aim #4 we will investigate how sortase attached cell wall proteins acquire heme iron from human hemoglobin during infections. Iron is an essential nutrient for bacterial growth and an understanding of heme capture at the molecular level could identify new targets for the development of antibiotics. Many of the steps in this process are performed by distantly related NEAT (NEAr iron Transporter) domains that have evolved distinct functions. Our research will study the first steps of heme iron capture, the binding of hemoglobin and heme by the NEAT domains in the IsdH and IsdC proteins, respectively. In particular, we will solve the first structure of a NEAT domain bound to heme (the IsdC-heme complex). (Lay Description) Bacteria infect humans using a variety of different proteins that they display on their surface. This research will study how these proteins are displayed and try to develop an inhibitor of this process that can be used as an antibiotic to treat bacterial infections.